Novel post-translationally cleaved Ara h 2 proteoforms: Purification, characterization and IgE-binding properties.

The 2S albumins Ara h 2 and Ara h 6 have been shown to be the most important source of allergenicity in peanut. Several isoforms of these allergens have been described. Using extraction and liquid chromatography we isolated proteins with homology to Ara h 2 and characterized hitherto unknown Ara h 2 proteoforms with additional post-translational cleavage. High-resolution mass spectrometry located the cleavage site on the non-structured loop of Ara h 2 while far UV CD spectroscopy showed a comparable structure to Ara h 2. The cleaved forms of Ara h 2 were present in genotypes of peanut commonly consumed. Importantly, we revealed that newly identified Ara h 2 cleaved proteoforms showed comparable IgE-binding using sera from 28 peanut-sensitized individuals, possessed almost the same IgE binding potency and are likely similarly allergenic as intact Ara h 2. This makes these newly identified forms relevant proteoforms of peanut allergen Ara h 2.

The structure and potential allergenicity of peanut allergen monomers after roasting.

Given that roasted peanut (Ro) products are commonly used in daily life, peanut allergenicity is a foremost concern. Analyzing the changes in the structure and potential allergenicity of individual allergens can promote the exploration of the structural basis of the alterations in the potential allergenicity of Ro. This work focused on four major allergens in raw peanut (Ra) and Ro. Structural changes were analyzed on the basis of circular dichroism, ultraviolet and fluorescence spectroscopy, and molecular dynamic simulation. The IgE recognition capability of allergens was assessed via western blot analysis. The IgE binding capacity of allergens was detected by conducting enzyme-linked immunosorbent assay. The potential allergenicity of allergens was evaluated using the KU812 cell degranulation model. The results showed that roasting induced different changes in the overall structures of allergens and altered the structures and electrostatic potential of IgE epitopes, especially Ara h 1 and Ara h 6. These alterations affected the potential allergenicity of allergens. Ara h 1 and Ara h 6 in Ro showed significantly enhanced IgE binding capacities and abilities to elicit KU812 cell degranulation, while Ara h 2 and Ara h 3 did not change significantly. For total protein, the roasted peanut protein showed decreased abilities to elicit KU812 cell degranulation. The results indicated that different allergens in Ro showed different changes of structures and potential allergenicity and that the conformational structure plays a crucial role in potential allergenicity of allergens.

Antioxidant Activities of Different Peanut (Arachis hypogaea L.) Market Types by Spectrophotometric Techniques Combined with Chemometrics.

Peanut is rich in oil and protein and has a large content of bioactive constituents consisting of tocopherols, phytosterols, and so on. Generally, Virginia, Spanish, Valencia and Runner market types are grown of peanut. In this study, it is aimed to determine the antioxidant activity, total phenolic content and total flavonoid content of peanuts from four different market types, for the first time, and group them with principal component analysis (PCA) and hierarchical cluster analysis (HCA). For PCA, PC1 and PC2 explained 87.655 % of the total variation and, according to the HCA of peanut samples, two main groups were determined. The total phenolic content changed 1.556 to 2.899 mg GAE/g. The lowest value have seen at Spanish merket type to determine the antioxidant activities of peanut samples were maked FRAP and DPPH assay, the lowest FRAP value (8.136 µmol FeSO47H2O/g sample) was seen at Valencia market type, the highest (14.004 µmol FeSO47H2O/g sample) was seen at Virginia market type. It was determined that the total flavonoid, total phenolic content, and antioxidant activities of the Virginia, Valencia, Spanish, and Runner market types included in the study were different from each other, and the Virginia market type showed superior characteristics compared to the others. The results obtained suggest that Virginia market type may be preferred more especially in peanut cultivation for food uses. It is thought that this study can be a source for future studies by eliminating a deficiency in the literature.

Mass Spectrometry Analysis on the Breakage of Allergens in High-Molecular-Mass Polymer of Roasted Peanuts.

Peanut allergy is a prevalent and concerning food allergy. Roasting can introduce structural changes to peanut allergens, affecting their allergenicity, but the structure on the primary structure is unclear. Here, the breakage sites were identified by mass spectrometry and software tools, and structural changes were simulated by molecular dynamics and displayed by PyMOL software. Results revealed that the appearance frequencies of L, Q, F, and E were high at the N-terminal of the breakage site, while S and E were dominant at the C-terminal. In the conformational structure, breakage sites were found close to disulfide bonds and the Cupin domains of Ara h 1 and Ara h 3. The breakage of allergens destroyed linear epitopes and might change the conformation of epitopes, which could influence peanuts' potential allergenicity.

Effects of Pretreatment on Stability of Peanut Oil Bodies and Functional Characteristics of Proteins Extracted by Aqueous Enzymatic Method.

Effects of dry and wet grind on peanut oil and protein yield, oil bodies (OBs) stability, fatty acid composition, protein composition and functional characteristics were systematically analyzed. Results showed that peanut oil and protein yields reached highest at dry grind 90 s (92.56% and 83.05%, respectively), while peanut oil and protein yields were 94.58% and 85.36%, respectively, at wet grind 120 s. Peanut oil and protein yields by wet grind was 2.18% and 2.78% higher than that of dry grind, respectively. Surface protein concentration (Г) and absolute value of zeta potential of OBs extracted by wet grind (WOBs) were 11.53 mg/m 2 and 18.51 mV, respectively, which were higher than OBs extracted by dry grind (DOBs), indicating stability of WOBs was higher than DOBs. Relative contents of oleic acid and linoleic acid in peanut oil, essential and hydrophobic amino acids in protein extracted by wet grind were higher than dry grind. There was little difference in protein composition between wet and dry grind, but thermal denaturation degree of protein obtained by wet grind was lower than dry grind. Solubility, oil retention, emulsion stability, foaming and foam stability of protein obtained by wet grind were better than dry grind. Results from this study provided theoretical basis for grind pretreatment selection of aqueous enzymatic method.

Atmospheric cold plasma reduces Ara h 1 antigenicity in roasted peanuts by altering the protein structure and amino acid profile.

Ara h 1 is the major allergen in peanuts. To enhance the unique flavor, peanuts are usually roasted at high temperatures. However, roasting can increase the allergenic potential, owing to glycation of allergens. Atmospheric cold plasma (ACP) is a non-thermal processing technology that generates reactive species, enabling protein structural changes. Herein, glucose was also added to the ACP-treated peanut protein before roasting. The content and antigenicity of the advanced glycation end products were measured. The antigenicity was evaluated by ELISA and in vitro digestion assays. The amino acid profile and secondary and tertiary protein structures were also assessed. The antigenicity of Ara h 1 decreased by 91 % and 76 % after 30 min of air and nitrogen plasma treatment, respectively. The glycation degree and thermal and digestive stabilities were also reduced. These results correlated with the structural changes, denaturation, and aggregation. Therefore, cold plasma may reduce the allergic effects of peanuts.

Facile and Ultrasensitive Food Allergen Quantification Using Microzone Paper-Based Mass Spectrometric Immunoassay.

Highly sensitive and rapid measurement of food allergens is essential to avoid unanticipated food allergies and to determine whether cross-contamination occurs in the food industry. Commercial immunoassay kits offer high specificity and convenience for allergen detection but still suffer limited quantitative sensitivity, accuracy, and stability based on the optical readout. In this work, a paper-based mass spectrometric immunoassay platform was constructed to achieve facile and highly sensitive quantification of peanut allergen, which combined the advantages of good specificity and accurate quantification from mass spectrometry and simplicity from a paper-based immunoassay. In this platform, a novel quaternary ammonium-based mass tag and a paper chip with a microzone were designed and developed, contributing to a large signal enhancement. This method was able to detect Ara h1 with a linear range of 0.1-100 ng mL-1 and a detection limit of 0.08 ng mL-1 in milk matrices. It has also been successfully applied to the accurate quantification of Ara h1 in six milk-related beverages, two biscuits, and two candy bars with complicated matrices and presented a low-concentration quantitation capability. This method gives a new type of mass spectrometric immunoassay for rapid and ultrasensitive allergen regulation in the food industry and for individual allergen differentiation research.

Screening of probiotic Lactobacillus to reduce peanut allergy and with potential anti-allergic activity.

BACKGROUND: Peanut is a significant source of nutrition and a valuable oilseed crop. It is also a serious allergy source, which poses a threat to 1.1% of the population. This study aimed to screen lactic acid bacteria (LAB) with the capacity to alleviate peanut allergenicity and exhibit anti-allergic properties. RESULT: The results show that LAB can make use of substances in peanuts to reduce the pH of peanut milk from 6.603 to 3.593-4.500 by acid production and that it can utilize the protein in peanuts to reduce the allergenic content (especially Ara h 1) and improve biological activity in peanut pulp. The content of Ara h 1 peanut-sensitizing protein was reduced by 74.65% after fermentation. The protein extracted from fermented peanut pulp is more readily digestible by gastrointestinal juices. The inhibitory activity assay of hyaluronidase (an enzyme with strong correlation to allergy) increased from 46.65% to a maximum of 90.57% to reveal that LAB fermentation of peanut pulp exhibited a robust anti-allergic response. CONCLUSION: The strains identified in this study exhibited the ability to mitigate peanut allergenicity partially and to possess potential anti-allergic properties. Lactobacillus plantarum P1 and Lactobacillus salivarius C24 were identified as the most promising strains and were selected for further research. © 2023 Society of Chemical Industry.

Investigation of peanut allergen-procyanidin non-covalent interactions: Impact on protein structure and in vitro allergenicity.

Interactions between plant polyphenols and food allergens may be a new way to alleviate food allergies. The non-covalent interactions between the major allergen from peanut (Ara h 2) with procyanidin dimer (PA2) were therefore characterized using spectroscopic, thermodynamic, and molecular simulation analyses. The main interaction between the Ara h 2 and PA2 was hydrogen bonding. PA2 statically quenched the intrinsic fluorescence intensity and altered the conformation of the Ara h 2, leading to a more disordered polypeptide structure with a lower surface hydrophobicity. In addition, the in vitro allergenicity of the Ara h 2-PA2 complex was investigated using enzyme-linked immunosorbent assay (ELISA) kits. The immunoglobulin E (IgE) binding capacity of Ara h 2, as well as the release of allergenic cytokines, decreased after interacting with PA2. When the ratio of Ara h 2-to-PA2 was 1:50, the IgE binding capacity was reduced by around 43 %. This study provides valuable insights into the non-covalent interactions between Ara h 2 and PA2, as well as the potential mechanism of action of the anti-allergic reaction caused by binding of the polyphenols to the allergens.

Electrochemical determination of the procyanidins in peanut skin using a carbon nanotube electrode.

We demonstrated the electrochemical detection of procyanidins in peanut skin, which is often a waste product of the food industry, using a carbon nanotube electrode. Procyanidins, the main ingredients of peanut skin, are oligomers of catechin or epicatechin; therefore, they have various forms such as dimers, trimers, and a different number of linkages between monomers. Quantification using traditional high-performance liquid chromatography-mass spectroscopy (HPLC-MS) is tedious, because many peaks can be traced. The use of CNT electrodes for procyanidin sensing is promising, because CNT's properties, such as high conductivity, catalytic ability, and special geometry (high ratio of surface area to volume), enable common and specific profiles of the cyclic voltammograms (CVs) of procyanidins. Furthermore, the intensity of the anodic peaks (+ 0.32 V) due to the oxidation of catechol groups is proportional to the concentration of procyanidin (linear rang: 2.8-88 mg L-1, sensitivity: 1.4 mA mg-1 L cm-2), and does not depend on the type of procyanidin. The amount of procyanidins in the peanut skin estimated by CV was similar to that estimated by HPLC-MS. This study may contribute to accelerating the utilization of peanut skin for animal food, drugs, and supplementation.

Effect of Electron-Beam Irradiation on Functional Compounds and Biological Activities in Peanut Shells.

Peanut shells, rich in antioxidants, remain underutilized due to limited research. The present study investigated the changes in the functional compound content and skin aging-related enzyme inhibitory activities of peanut shells by electron-beam treatment with different sample states and irradiation doses. In addition, phenolic compounds in the peanut shells were identified and quantified using ultra-performance liquid chromatography with ion mobility mass spectrometry-quadrupole time-of-flight and high-performance liquid chromatography with a photodiode array detector, respectively. Total phenolic compound content in solid treatment gradually increased from 110.31 to 189.03 mg gallic acid equivalent/g as the irradiation dose increased. Additionally, electron-beam irradiation significantly increased 5,7-dihydroxychrome, eriodictyol, and luteolin content in the solid treatment compared to the control. However, liquid treatment was less effective in terms of functional compound content compared to the solid treatment. The enhanced functional compound content in the solid treatment clearly augmented the antioxidant activity of the peanut shells irradiated with an electron-beam. Similarly, electron-beam irradiation substantially increased collagenase and elastase inhibitory activities in the solid treatment. Mutagenicity assay confirmed the stability of toxicity associated with the electron-beam irradiation. In conclusion, electron-beam-irradiated peanut shells could serve as an important by-product with potential applications in functional cosmetic materials.

Target-Induced Electrochemical Sensor Based on Foldable Aptamer and MoS2@MWCNTs-PEI for Enhanced Detection of AFB1 in Peanuts.

Herein, a sensitive and selective electrochemical sensor based on aptamer folding was constructed to detect aflatoxin B1 (AFB1) in peanuts. Specifically, polyethylenimine-functionalized multiwalled carbon nanotubes modified with molybdenum disulfide (MoS2@MWCNTs-PEI) were used as the electrode matrix to enable a large specific surface area, which were characterized by the Randles-Sevcik equation. Additionally, AuNPs were used to immobilize the aptamer via the Au-S covalent bond and provide a favorable microenvironment for signal enhancement. Methylene blue (MB) was modified at the proximal 3' termini of the aptamer as the capture probe, while the signal transduction of the sensor was obtained through changes in conformation and position of MB induced by the binding between AFB1 and the probe. Changes in spatial conformation could be recorded by electrochemical methods more readily. This electrochemical aptasensor demonstrated remarkable sensitivity to AFB1 with an extensive detection range (1 pg/mL to 100 ng/mL) and a lower limit detection (1.0 × 10-3 ng/mL). Moreover, using the constructed aptasensor, AFB1 was identified successfully in peanut samples, with recoveries ranging from 95.83 to 107.53%, illustrating its potential use in determining AFB1 in food.

Allelopathic Mechanisms in Camellia oleifera-Arachis hypogaea L. Intercropping.

Tree-crop intercropping is of great significance in food security, land protection, and sustainable agriculture. However, the mechanisms of allelopathy between plant species during intercropping are still limited. This study focuses on the allelopathic effects in the intercropping between Camellia oleifera and Arachis hypogaea L. in southern China. We use different parts of the C. oleifera extract to evaluate their impact on peanut seed germination. The results showed that it has inhibitory effects on peanut germination and growth, with the fruit shell having the strongest inhibitory effect. Three main allelopathic substances affecting A. hypogaea germination and growth were identified using gas chromatography-mass spectrometry (GC-MS) analysis, namely, 2,4-di-tert-butylphenol, hexanal, and benzaldehyde. Transcriptomics and metabolomics analyses revealed their effects on glutathione metabolism pathways and specific gene expression. In summary, this study reveals the allelopathic interaction mechanism between C. oleifera and A. hypogaea, which helps to better understand the role of allelopathy in intercropping practices between trees and crops.

Green process for the preparation of resveratrol-containing high oleic acid peanut oil.

Resveratrol (Res), a polyphenol compound with strong biological activity, is widely used in medicinal and health products. In this study, an innovative resveratrol high oleic peanut oil (Res-HOPO) was prepared utilizing self-developed cold pressing equipment and high oleic peanuts. The peanut roots were pretreated with four different methods, including ultra-fine crushing, ultrasound-treating, microwave-treating, and a combination of microwave-ultrasound-treating peanut roots. Under optimized conditions (microwave power of 15 W, ultrasound time of 28 min, and ultrasound power of 400 W), the Res-HOPO prepared by pretreating with a combination of microwave-ultrasound had the most Res (91.12 mg/kg). Except for the pretreated whole peanut roots, pretreating with microwave (40.49 mg/kg), ultrasound (39.03 mg/kg), and ultra-fine crushing of peanut root powder (22.57 mg/kg) resulted in the high Res content. The Res-HOPO had a satisfactory yield (40%), oleic acid content (74.05% ∼ 75.85%), no trans fatty acids, great physicochemical properties, higher nutritional value (4-fold increase in squalene and almost 10-fold increase in campesterol), an extended oxidation induction time (1.39 âˆ¼ 22 times), and no heavy metals, pesticides, or aflatoxins. The four green pretreatment methods used for the preparation of Res-HOPO in this study were effective, which provided an innovative approach for developing nutritious and healthy edible oil.

Changes in the structure and hydration properties of high-temperature peanut protein induced by cold plasma oxidation.

To improve the hydration properties of high-temperature pressed peanut protein isolate (HPPI), we investigated the effect of cold plasma (CP) oxidation on functional and structural properties. Compared to HPPI, the hydrated molecules number and the surface contact angle were significantly decreased at 70 W, from 77.2 × 109 to 17.7 × 109 and from 85.74° to 57.81°, respectively. The reduction of the sulfhydryl content and the increase of the disulfide bond and di-tyrosine content indicated that the structural transformation was affected by the oxidation effect. In terms of structural changes, a stretched tertiary structure, ordered secondary structure, and rough apparent structure were observed after CP treatment. Additionally, the enhancement of surface free energy and group content such as -COOH, -CO and -OH on the surface of HPPI contributed to the formation of hydrated crystal structures. In general, the oxidation effect of CP effectively improved the hydration properties of HPPI and broaden its application field.

Kaolinite reduced Cd accumulation in peanut and remediate soil contaminated with both microplastics and cadmium.

Microplastics (MPs) increase the effective state of heavy metals (HMs) in soil and seriously threaten the yield and quality of peanuts (Arachis Hypogea L.). Kaolinite (KL) has the potential to ameliorate MP- and HM- contaminated soils, but the mechanism of action between them is not well understood. Therefore, 60-day experiments were conducted, where KL (1 %, 2 %) and MPs (0.1 %, 1 %) were individually or jointly mixed into soils with different cadmium (Cd) concentrations (0.5, 2.5, and 5.0 mg·kg-1) to cultivate peanuts in a greenhouse. Finally, soil-bioavailable Cd, peanut dry weight, peanut Cd concentrations, the pH, cation exchange capacity (CEC), dissolved organic carbon (DOC), microbial biomass carbon (MBC) and microbial biomass nitrogen (MBN) were determined. It was shown that MPs negatively affected the peanut dry weight and increased the content of soil-bioavailable Cd and Cd concentration in peanut. In the MP- and Cd-contaminated soils, KL mitigated the negative influence of MPs by increasing the dry weight of peanuts by 8.40 %-40.59 %, decreasing the soil-bioavailable Cd by 23.70-35.74 %, and significantly decreasing peanut Cd concentrations by 9.65-30.86 %. The presence of MPs decreased soil pH (7.69-7.87) and the CEC (20.96-23.95 cmol·L-1) and increased the soil DOC (1.84-2.26 mg·kg-1). KL significantly increased soil pH (7.79-8.03) and the CEC (24.96-28.28 cmol·L-1) and mitigated the adverse influence of MPs on the pH and CEC of Cd-contaminated soils. A regression path analysis (RPA) evidenced that KL decreased Cd accumulation in plants by changing the properties of soil contaminated with MPs and Cd. The research results revealed the mechanism of KL on peanut growth and Cd absorption in MP- and Cd-contaminated soil. The results of this study provide a foundation to improve the quality of MP- and HM-contaminated soils and realize safe peanut production.

Quantitative analysis of aflatoxin B1 of peanut by optimized support vector machine models based on near-infrared spectral features.

This study designs a chemometric framework for quantitatively evaluating aflatoxin B1 (AFB1) in peanuts based on near-infrared (NIR) spectroscopy technique. The NIR spectra of peanut samples exhibiting diverse fungal contamination levels were acquired using a portable NIR spectrometer. Subsequently, appropriate pre-processing techniques were employed for data refinement. To streamline the analysis, the iterative variable subset optimization (IVSO) technique was employed to conduct an initial screening of the pre-processed NIR spectra, eliminating numerous irrelevant variables. Building upon this screening process, the beluga whale optimization (BWO) algorithm was utilized to optimize the selected feature variables further. Subsequently, support vector machine (SVM) models were developed using the refined near-infrared spectral features to test AFB1 in peanuts quantitatively. The results indicate that the SVM model significantly improves detection performance and generalization proficiency, particularly after secondary optimization using BWO-IVSO. Among the different models considered, the SVM model established after BWO-IVSO optimization exhibited the most extraordinary level of generalization, with a root mean square error of prediction of 24.6322 µg∙kg-1, a correlation coefficient of 0.9761, and a relative percent deviation of 4.6999. Overall, this investigation highlights the effectiveness of the proposed NIR spectroscopy model based on BWO-IVSO-SVM for quantitatively analyzing AFB1 in peanuts. The study contributes valuable technical and methodological insights that can serve as a reference for rapidly determining mycotoxins in cereal crops.

A novel whole peanut butter refined by stirred media mill: The size, microstructure, rheology, nutrients, and flavor.

A novel whole peanut butter (PB) was developed using an emerging technology called stirred media mill (SMM). The impact of SMM on the size, microstructure, rheology, nutrient, and flavor of PB was investigated. The SMM treatment significantly decreased the particle size of PB, damaged cell structure, and released the oil body from cells. The apparent viscosity of PB decreased with the grinding process. Visual inspection revealed that the colloidal stability of PB was improved. The fatty acid composition was not affected by the grinding process. However, the tocopherol contents of the extracted oil slightly increased. Electronic nose and GC-MS analysis indicated that SMM could alter the flavor of PB after grinding for 45 min. Overall, SMM was a potential process technology to manufacture stable nut butter with smooth texture and delightful flavor profile.

Valorization of Peanut Skin as Agricultural Waste Using Various Extraction Methods: A Review.

Peanuts (Arachis hypogea) can be made into various products, from oil to butter to roasted snack peanuts and candies, all from the kernels. However, the skin is usually thrown away, used as cheap animal feed, or as one of the ingredients in plant fertilizer due to its little value on the market. For the past ten years, studies have been conducted to determine the full extent of the skin's bioactive substance repertoire and its powerful antioxidant potential. Alternatively, researchers reported that peanut skin could be used and be profitable in a less-intensive extraction technique. Therefore, this review explores the conventional and green extraction of peanut oil, peanut production, peanut physicochemical characteristics, antioxidant activity, and the prospects of valorization of peanut skin. The significance of the valorization of peanut skin is that it contains high antioxidant capacity, catechin, epicatechin resveratrol, and procyanidins, which are also advantageous. It could be exploited in sustainable extraction, notably in the pharmaceutical industries.

9-Oxononanoic Acid and Its Lysine Schiff Base Adduct as a Novel Lipation Product in Peanuts.

9-Oxononanoic acid (9-ONA) was quantitated in peanuts roasted at 170 °C by GC-MS (EI). After roasting peanuts for 40 min, 9-ONA decreased from 1010 µmol/kg protein in the unheated sample to 722 µmol/kg protein, most likely due to modifications of nucleophilic side chains of protein-bound amino acids (lipation). After heating Nα-acetyl-l-lysine and 9-ONA in model experiments, a Schiff base in its reduced form, namely, Nε-carboxyoctyl-acetyl lysine, as well as two isomeric pyridinium derivatives, namely, dicarboxyhexylcarboxyheptylpyridinium-acetyl lysine 1 and 2, were tentatively identified by HPLC-ESI-MS/MS. Based on the identified lipation products of 9-ONA, it can be assumed that lipation reactions represent a mirror-image reaction. For quantitation of Nε-carboxyoctyllysine (COL) in roasted peanuts by means of HPLC-ESI-MS/MS, samples were reduced with sodium borohydride and acid hydrolyzed. For the first time, COL was quantitated after reduction in roasted peanuts. Furthermore, after prolonged roasting of peanuts for 40 min, COL decreased from 139.8 to 22.5 µmol/kg protein, which provides initial evidence for lipation of nucleophilic side chains of protein-bound amino acids by glycerol-bound oxidized fatty acids (GOFAs, e.g., 9-ONA) with the formation of neo-lipoproteins.